Peanut (Arachis hypogaea) sprout prevents high-fat diet-induced cognitive impairment by improving mitochondrial function

This study was performed to evaluate the improvement effect of the ethyl acetate fraction from peanut (Arachis hypogaea) sprout (EFPS) on high-fat diet (HFD)-induced cognitive deficits in C57BL/6 mice. Mice were randomly divided four groups (n = 13) as control (normal chow), HFD, EFPS 20 (20 mg/kg of body weight; intragastric administration) and EFPS 50 (50 mg/kg of body weight; intragastric administration) groups. HFD was provide for 15 weeks excepting control group. EFPS ameliorated cognitive dysfunction in Y-maze, passive avoidance test and Morris water maze test. EFPS significantly improved glucose tolerance and serum lipid profile, and reduced body weight. EFPS ameliorated oxidative stress by regulating MDA levels and SOD activity in liver and brain tissues. In addition, EFPS restored brain mitochondrial dysfunction related to energy metabolism. Moreover, the bioactive compounds of EFPS were identified as di-caffeic acid, caffeic acid, dihydrokaempferol-hexoside, di-p-coumaroyl tartaric acid isomer and group B soyasaponins using ultra-performance liquid chromatography-quadrupole-time-of-flight (UPLC-Q-TOF) mass spectrometry. These results show that EFPS can improve cognitive functions in HFD-induced diabetic mice.

Behavioral tests. Y-maze, passive avoidance, and MWM tests were performed to estimate the effect of EFPS on cognitive function. In the result of Y-maze test, the number of arm items was no significant difference between all groups (Fig. 2a). The alternation behavior of the HFD group (49.85%) decreased compared with the control group (76.03 ± 8.59%). However, the EFPS groups (EFPS 20; 76.16 ± 5.62% and EFPS 50; 75.47 ± 4.67%) effectively improved alternation behavior compared with the HFD group.
In the results of passive avoidance test, the HFD group had lower step-through latency (66.67 ± 65.26 s) in retention than the control groups (Fig. 2b). However, the EFPS-treated groups had higher step-through latency (EFPS 20; 123.75 ± 69.45 s and EFPS 50; 282.00 ± 36.00 s) in retention compared to the HFD group.
The MWM test was performed to evaluated spatial learning and long-term memory function, and the results are shown in Fig. 2c-d. There were differences in escape latency among each of the groups on the first day. The latency in finding the waterlogged platform of all groups decreased every day, but HFD group was significantly longer than the other groups. Case of probe test, the HFD group showed a 33.60 ± 5.67% decrease in the time spent in the quadrant with the platform (Fig. 2d). Compared to the HFD group, the time spent in the quadrant with the platform of the EFPS 50 group increased significantly by 51.17 ± 11.20%. Serum biochemicals. The levels of serum biochemicals are shown in Tables 2 and 3. The levels of TCHO, TG and LDLC were remarkably high in the HFD group (236.89 ± 33.39 mg/dL, 154.58 ± 17.72 mg/ dL and 39.44 ± 2.61 mg/dL) compared to the control group (110.20 ± 7.73 mg/dL, 109.60 ± 11.24 mg/dL and 16.28 ± 6.11 mg/dL) ( Table 2). However, these biomarkers levels decreased in the EFPS groups (EFPS20; 198.00 ± 47.63 mg/dL, 137.50 ± 18.64 mg/dL, and 29.96 ± 6.57 mg/dL, EFPS 50; 198.75 ± 17.33 mg/dL, 126.00 ± 28.19 mg/dL and 22.84 ± 2.96 mg/dL). Additionally, HTR (%) showed higher levels in the EFPS groups (EFPS 20; 70.36 ± 6.67% and EFPS 50; 75.03 ± 5.56%) compared to the HFD group (66.49 ± 9.27%).
Hepatic and renal toxicity was confirmed by serum GOT, GPT, BUN, CRE and LDH levels ( Table 3). HFD consumption (169.20 ± 19.40 U/L, 243.40 ± 36.18 U/L, 15.94 ± 1.10 mg/dL and 722.14 ± 72.66 U/L) caused an increase in GOT, GTP, LDH, and BUN levels compared to the control group (50.40 ± 1.52 U/L, 33.60 ± 3.05 U/L, 265.80 ± 67.86 U/L and 13.70 ± 1.43 mg/dL). EFPS treatment (EFPS 20; 111.00 ± 7.78 U/L, 151.20 ± 33.39 U/L, 427.20 ± 62.28 U/L and 14.54 ± 0.62 mg/ dL, EFPS 50; 90.40 ± 6.11 U/L, 98.40 ± 12.42 U/L, 429.20 ± 67.75 U/L and 14.58 ± 1.08 mg/dL) lowered the level of GOT, GTP, BUN and LDH compared to the HFD group. There was no significant difference in serum CRE level between all groups. Antioxidant effect. The protective effect of EFPS against oxidative stress in liver and brain tissue was confirmed by measuring MDA contents and SOD level (Fig. 3). The MDA contents in the brain (4.50 ± 0.40 nmole/ mg of protein) and liver (6.81 ± 0.85 nmole/mg of protein) tissues of the HFD group increased compared to the control group (3.99 ± 0.58 and 3.45 ± 0.76 nmole/mg of protein) (Fig. 3a,b). However, the hepatic MDA contents showed decreased in the EFPS 50 group (cerebral tissue; 4.45 ± 0.31 and hepatic tissue; 5.72 ± 0.41 nmole/mg of protein) compared to the HFD group. There is no significant difference between EFPS 50 group and HFD group on cerebral MDA contents. Table 1. Effect of ethyl acetate fractions from peanut (Arachis hypogaea) sprout (EFPS) on organ weight change (Unit: g). The results were shown with mean ± SD (n = 5). Data were statistically considered at p < 0.05, and different small letters represented a statistical difference.    www.nature.com/scientificreports/ Mitochondrial parameters. The effect of EFPS on brain mitochondrial dysfunction was assessed by measuring ROS production, MMP and ATP contents (Fig. 4). The mitochondria ROS production was measured on using DCF-DA (Fig. 4a). The ROS production in the HFD group increased fluorescence intensity (167.71 ± 16.83%) more than the control group (100.00 ± 1.52% Neuronal apoptosis. To confirm the effect of EFPS on apoptosis in brain tissue, the relative expression of proteins detected, and shown on Fig. 5. As the result of B-cell lymphoma 2 (Bcl-2), the expression level was decreased in the HFD group (0.58 ± 0.19) compared to the control group (1.00 ± 0.16). In addition, the HFD group showed increased expression level of Bcl-2 associated X (Bax) (1.27 ± 0.05). However, the EFPS 50 group significantly improved on Bcl-2 (1.04 ± 0.10) and Bax (0.74 ± 0.19) compared to the HFD group.
Bioactive compounds analysis. The major bioactive compounds of EFPS were detected using a liquid  www.nature.com/scientificreports/

Discussion
Recent growing evidence demonstrates an association with obesity and cognitive impairment 15 . Chronic HFD intake induces obesity, T2DM, and impairment of short-term, working, and long-term memory 16 . In addition, the long-term consumption of HFD damages brain function via inflammatory response, insulin resistance, oxidative stress, and induction of mitochondrial dysfunction 17 . Therefore, the present study was conducted to assess the protective effect of EFPS on cognitive impairment caused by HFD. Chronic HFD consumption increases free fatty acids decomposition, which can lead to an increase in very low-density lipoprotein (VLDL) and TG in blood 18 . In the hyperlipidemia state caused by HFD, insulin is involved www.nature.com/scientificreports/ in the production or removal of TG, causing metabolic disorders 19 . Insulin reduces hepatic gluconeogenesis and glycogenolysis and promotes TG storage in adipocytes, thereby reducing blood sugar level. However, chronic HFD intake induces insulin resistance in liver tissue, which directly interferes with glucose metabolism 20,21 . In this study, hyperglycemia and insulin resistance were induced in the HFD group (Table 1 and Fig. 1). However, EFPS restored insulin resistance by improving fasting blood glucose and impaired glucose tolerance. With these effects it is considered that EFPS improve insulin sensitivity and normalized blood glucose levels by reducing the accumulation of fat and suppressing abnormal blood lipid metabolism. In previous studies, group B soyasaponins were reported to improve the lipid profile by lowering TCHO, TG, LDLC and free fatty acids in serum 22 . In addition, they reduced hepatic TG accumulation by down-regulating the mRNA expression of sterol regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) related to lipogenesis 23 . Considering these factors, group B soyasaponins might improve obesity-related metabolic dysfunction with hepatic insulin resistance in HFD mice. Therefore, it is considered that EFPS, which is rich in soyasaponin, might assist in the prevention of obesity and diabetes caused by HFD. www.nature.com/scientificreports/ High glucose levels due to insulin resistance, which are derived early in T2DM, affect the brain 24 . In addition, high glucose levels damage the vulnerable brain from oxidative stress, causing impairment of learning and memory function in diabetic patients 25,26 . Previous studies also proved that HFD could cause cognitive impairment 27,28 . In this study showed that HFD induced cognitive deficits in mice. However, EFPS improved learning and memory function impairment in the Y-maze, passive avoidance and Morris water maze behavioral tests (Fig. 2). Bioactive compounds such as caffeic acid, di-p-coumaroyl tartaric acid and group B soyasaponins were identified in EFPS (Table 4, Fig. 6). Caffeic acid improves memory impairment by ameliorating cerebral insulin and leptin signaling pathways in HFD-induced rats 29 . According to Hong et al. 30 , soyasaponin Bb protected learning and memory deficits by supporting neuronal regeneration processes and inhibiting hippocampal inflammation in cognitive impairment model rats. Moreover, it has been reported that soyasaponin Bb improves memory ability by increasing brain-derived neurotrophic factor (BDNF) protein expression in scopolamineinduced cognitive deficient mice 31 . In conclusion, it is considered that EFPS including various bioactive compounds considerably restored cognitive dysfunction.
The hyperglycemic state accelerates ROS production, lipid peroxidation oxidative modifications of DNA and proteins and in various tissues 32,33 . Especially, hepatic oxidative stress might interfere the insulin-signaling cascade and disturb with the normal transcription of certain proteins, induces insulin resistance 34 . Oxidative stress can cause liver cell damage and steatohepatitis by producing excessive oxidants and cytokines 35 . Also, the consequently increased ROS and reduced anti-oxidant activity, leading to damage on vascular cells, Schwann cells, neurons, as well as multiple other organs 36 . In particular, continuous hyperglycemia due to various causes such as obesity, promotes the production of ROS in brain tissue vulnerable to oxidative stress 37 . Brain tissue damaged by ROS and oxidative stress affects cognitive dysfunction in AD patients 38 . Based on previous studies, the antioxidant activities of liver and brain tissues were evaluated by measuring MDA content and SOD levels  Table 4. Identification of main compounds in the ethyl acetate fraction from peanut (Arachis hypogaea) sprout (EFPS) by UPLC-Q-TOF/MS E system. www.nature.com/scientificreports/ in this study. Consequently, EFPS reduced hepatic and brain oxidative stress caused by hyperglycemia (Fig. 3). p-Coumaric acid reduced HFD-induced oxidative stress by increasing antioxidant system in serum and liver tissues 39 . Moreover, it has been reported that consumption of caffeic acid improved the antioxidant system in the brain tissue of lipopolysaccharide (LPS)-induced neuroinflammation mice 40 . According to Salau et al. 41 , caffeic acid improved antioxidant enzymes by increasing glutathione, SOD, and catalase activities in brain tissues. These results suggest that EFPS can prevent damage to beta cells of the pancreas by protecting oxidative stress in the liver, and consequently contribute to the improvement of insulin resistance. EFPS is thought to protect liver tissue by activating the antioxidant system in the liver after damage induced by HFD. In addition, EFPS might improve cognitive dysfunction by protecting the antioxidant system in the brain against HFD-induced cytotoxicity. The brain mitochondria play an important role by regulating calcium homeostasis, an important mechanism for energy demanding neurotransmission and learning and memory processes 42 . Also, the mitochondria contribute cellular energy generation in the form of ATP and maintain the balance between ROS production and detoxification 43 . However, excessive ROS production induces a variety of pathological conditions through oxidative damage in the brain 7,44 . Furthermore, when the formation of ROS is enhanced, it can impair mitochondrial function and the viability of cells 45 . Oxidative stress caused by high glucose damages mitochondria with rapid depolarization of MMP and generates more ROS through activation of inter-mitochondrial signaling networks 46 . Functional and structural of mitochondria degrades ATP production capability, which can lead to neurodegenerative diseases caused by mitochondrial disorders 6 . Previous studies have reported that consumption of HFD induces insulin resistance, which increases lipid peroxidation and oxidative stress. In addition, the excessive oxidative stress increases mitochondria membrane permeability, which promotes the secretion of apoptosis mediators such as Bcl-2 family and caspase 47 . Neuronal apoptosis is leads to neurodegeneration, ultimately leading to cognitive impairments such as AD. Therefore, many studies have been focused on finding agents that improve mitochondrial function, mainly based on the inhibition of oxidative stress. In the results of studying the protective effect of EFPS on mitochondrial deficit, EFPS significantly ameliorated mitochondrial dysfunction by restoration of mitochondrial ROS, MMT and ATP levels (Fig. 4).

Compound Retention time (min) Molecular formula Precursor ion [M-H] − Product ions
A mitochondrial dysfunction such as decrease in MMP, increase of ROS, and depletion of ATP leads to apoptosis in neuronal cells 49 . Apoptosis of the intrinsic pathway is mostly inhibited or activated by Bcl-2 family proteins, and bcl-2 and bax proteins are known as representative cytotoxic mediators 48 . Cytosolic Bax combined with Bcl-2 in mitochondrial outer membrane and releases cytochrome c indicating caspase cascade by various stress such as DNA damage and ROS production. This process induces apoptosis and indicate the initiation of mitochondrial damage. In this study, the EFPS presented the regulation effect of protein expression related to apoptosis indicator (Fig. 5). The caffeic acids identified in EFPS reportedly have mitochondrial protective effects in relation to their antioxidant effects 50 . p-Coumaric acid upregulated the expression of nuclear respiratory factor 1 (NRF-1), a transcription factor that is a key mediator of genes involved in mitochondrial biosynthesis 51 . According to Ueda et al. 52 , p-coumaric acid improved mitochondrial dysfunction by controlling neuronal cell apoptosis caused by insoluble mutant copper-zinc SOD-1 aggregation on N2a cells (a mouse neuroblasts). It was also reported that p-coumaric acid protects neurodegeneration by regulation Bcl-2 and Bax proteins in hippocampal tissue in T2DM mice 53 . Based on these studies, it is considered that the mitochondrial-related apoptosis protein regulated effect of EFPS is due to the neuroprotective effect of various bioactive compounds contained in EFPS. These findings suggest that EFPS is considered a potential material for HFD-induced cognitive dysfunction by suppressing mitochondrial dysfunction.
In conclusion, this study showed that EFPS ameliorated serum lipid biomarkers and learning and memory function deficits by attenuating antioxidant system damage and brain mitochondrial dysfunction in HFDinduced diabetic mice. Thus, EFPS could be effective for a therapeutic strategy to improve neurodegenerative diseases such as AD caused by long-term HFD consumption. However, more studies are required to identify the detailed mechanism of EFPS on the role of mitochondrial function between HFD-induced insulin resistance and cognitive decline. Sample preparation. All methods were performed in accordance with the relevant guidelines and regulations. Peanut sprouts were supplied by Hansaeng Bio Co. Ltd. (Korea) in May 2017 and complied with relevant institutional, national, and international guidelines and legislation. The cultivation conditions of peanut sprouts are as follows. Peanuts cultivated in Gochang-gun (Korea) were verified from National Institute of Forest Science (Suwon, Korea). The obtained peanuts were dried in sunlight to lower the moisture content under 6-10%, and then dried peanuts were shelled. Peanut sprouts were obtained by repeating watering process at intervals of 5 h in a dark room at 26 °C for 7 days, and dried using hot air drying at 60 °C for 20 h (Lassele DY-220H, Busan, Korea). This incubated peanut sprouts were extracted in 50-fold 80% ethanol for 3 h at 40 °C. The extract was filtered and concentrated using a rotary evaporator (N-N series, Eyela Co., Tokyo, Japan). Then, the obtained concentrate was resuspended distilled water, and successively fractionated with n-hexane, chloroform and ethyl www.nature.com/scientificreports/ acetate. Through a preliminary test, the ethyl acetate fraction from peanut (Arachis hypogaea) sprout (EFPS) was used as an optimal extraction condition (Figs. S1 and S2). EFPS was lyophilized and powdered, and stored at -20 °C until use.

Materials and methods
Animals and experimental design. All animals were maintained and used in accordance with ARRIVE guidelines 54 and all experimental procedures were approved according to guidelines established from the Institutional Animal Care and Use Committee of Gyeongsang National University (Certificate No. GNU-161116-M0065) on November 22, 2016. Male C57BL/6 mice (4 weeks old) were purchased from Samtako (Osan, Korea). Experiment condition was maintained under controlled temperature (22 ± 2 °C) and humidity (55%) with a 12 h light, 12 h dark cycles. The mice were randomly divided into 4 groups (n = 13; 8 for in vivo and ex vivo tests; 5 for mitochondrial tests), the control group was fed a normal diet, and the other groups were fed HFD for 15 weeks. To select the mice having hyperglycemia, the mice with a blood glucose level of 400 mg/mL or higher were selected, and randomly assigned to 3 mice per cage using the standard = RAND() function in Microsoft Excel. After, the EFPS dissolved in drinking water was intragastrically administrated to EFPS group (EFPS 20 and 50, 20 and 50 mg/kg of body weight, respectively) for 4 weeks. Control and HFD groups were provided by oral gavage the same dose of drinking water.

Intraperitoneal Glucose Tolerance Test (IPGTT).
After mice were fasted overnight, d-glucose (2 g/kg of body weight) dissolved in 0.85% NaCl was intraperitoneally administered. Blood glucose was measured at 0, 15, 30, 60, 90 and 120 min using an Accu-Chek glucose meter (Roche Diagnostics, Basel, Switzerland) by taking blood from the tail of mice.

Behavioral tests. Y-maze test.
Mice were placed in a white construct consisting of a Y-maze with equal angles between three arms, and recorded for each mouse for 8 min. The movements of each mouse were recorded and evaluated using a video tracking system (SMART v3.0; Panlab, Barcelona, Spain) 55 .
Passive avoidance test. The passive avoidance apparatus made of lighted and darked chamber, separated by a central manual door. For the training trial, mice were initially placed in the lighted chamber for 1 min and then the lights were turned on for 2 min. The central separation door was opened, and when mice entered the dark chamber, electrical foot shock (0.5 mA) of 3 s was given to mice through the stainless rods. After 24 h, the latency time to re-enter the dark chamber was recorded for a maximum of 300 s 55 .
Morris water maze (MWM) test. MWM pool consist of a stainless steel circular pool randomly divided into four sections (E, W, S and N) of equal area. The pool was filled with water dissolved in white non-toxic ink, and water temperature was maintained 20 ± 2 °C. A white platform was placed in the W zone and submerged. The test was conducted for 6 days, and contained three parts of the visible platform test (day 1), the hidden platform test (day 2-5), and the probe test (day 6). The visible platform test was conducted with the platform visible on the water surface for 1 day. Hidden platform test was conducted for 4 days, when a mouse did not locate the platform within 60 s, it was placed to the platform and left for 20 s. In the probe test, the platform was removed and the retention time of each mouse in the W zone was recorded during 90 s 56 . All behavior tests were conducted in blinded condition with no information to investigators.
Preparation serum and organs. After behavioral tests, the mice were sacrificed with CO 2 . Blood samples were collected from the postcaval vein and stored in heparin tube on ice. The brain, liver, spleen, kidney, white epididymal adipose and testis tissues were removed, rinsed with a saline and immediately weighed, and stored at − 70 °C. After the blood was centrifuges at 10,000g for 10 min at 4 °C, supernatant (i.e., serum) was immediately placed on ice and used for biochemical analysis. Antioxidant assay. Malondialdehyde (MDA) contents. To determine MDA contents, the brain and liver tissues were homogenized with phosphate-buffered saline (PBS) and centrifuged at 2500g for 10 min. The supernatants were reacted with 1% phosphoric acid and 0.67% TBA before they were incubated at 95 °C for 1 h. The absorbance was measured at 532 nm on microplate reader (Epoch 2, BioTek Instruments Inc., Winooski, VT, USA). The protein concentration was measured by the Bradford assay 58 .

SOD level.
To determine SOD level, PBS was added of homogenized brain and liver tissues and centrifuged to obtain a pellet. Cell extraction buffer (10% SOD buffer, 0.4% Triton X-100, and 200 μM phenylmethane sulfonylfluoride) was added and then placed on ice for 30 min. Then, the extract was centrifuged at 10,000g for 10 min, and the supernatant was used for a SOD analysis. SOD level was determined according to the manufacturer's protocol (Dojindo Molecular Technologies). www.nature.com/scientificreports/ Mitochondrial assay. Isolation of mitochondria. Brain mitochondria were isolated from whole brain tissue homogenized in the isolation buffer (1 mM EGTA, 0.1% BSA, 215 mM mannitol, 75 mM sucrose, 20 mM HEPES (Na + ) and 75 mM sucrose, pH 7.2) and centrifuged at 1300g for 5 min. The supernatant was centrifuged at 13,000g for 10 min again. The pellet was mixed with the isolation buffer containing 0.1% digitonin and 1 mM EGTA, and incubated for 5 min. Then, centrifuged at 13,000g for 10 min. The obtained pellets were resuspended in isolation buffer and re-centrifuged. The final mitochondrial pellets were suspended in isolation buffer without EGTA and used for mitochondrial analysis 59 .
ROS production. The mitochondrial ROS production was measured using a respiration buffer (500 μM EGTA, 2 mM potassium phosphate, 20 mM HEPES, 125 mM KCl, 2.5 mM malate, 5 mM pyruvate and 1 mM MgCl, pH 7.0). A mitochondrial extraction was incubated with 25 μM DCF-DA in respiration buffer at room temperature. After 20 min, the fluorescence intensity was measured using fluorometer at excitation wave 530 nm and emission wave 590 nm (Infinite 200, Tecan Co., San Jose, CA, USA) 59 .
The mitochondrial membrane potential (MMP). MMP was measured using the isolated mitochondria with a JC-1. The assay buffer contained mitochondrial isolation buffer with 5 mM malate and 5 mM pyruvate. The assay buffer and mitochondrial extraction were mixed on the 96-well black plate, addition of 1 μM JC-1. The fluorescence intensity was measured after 20 min (excitation; 530 nm and emission; 590 nm) 59 .
ATP contents. The ATP contents were determined on a luminescence meter (GloMax-Multi, Promega Corp.) using a kit (Promega Corp.) and calculated according a standard curve.
Western blot assay. Brain tissues were homogenized with cold lysis buffer (ProtinEx Animal cell/tissue, GeneAll Biotechnology, Seoul, Korea) containing 1% protease inhibitor cocktails (Thermo Fisher Scientific, Rockford, IL, USA). And then, homogenates were centrifuged at 13,000g at 4 °C for 10 min. The supernatant was quantified by Bradford reagent (Bio-Rad, CA, USA) assay 58 . The samples were loaded on a 12% polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After transfer, the membrane blocked by 5% skim milk, and reacted overnight in tris-buffered saline containing 0.1% Tween 20 (TBST) including each diluted primary antibody (dilution 1:1000) at 4 °C. Then, the membrane was washed twice with TBST and incubated with secondary antibody (dilution 1: 3000) for 1 h at room temperature. The washed membrane was detected with iBright CL1000 imager (Thermo Fisher Scientific, Waltham, MA, USA). The quantitative analysis of protein expression was performed by using Image J software (National Institutes of Health, Bethesda, MD, USA).
Bioactive compounds analysis. The bioactive compound analysis of EFPS were analyzed using a UPLC-Q-TOF/MS E . A UPLC system (Vion, Waters Corp., Milford, MA, USA) was used and analytical samples were injected into an Acquity UPLC BEH C 18 column (100 mm × 2.1 mm × 1.7 µm) (Waters Corp.). Gradient condition was conducted as 0.1% formic acid in distilled water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) at a flow rate of 0.35 mL/min for 12 min. The gradient condition of mobile phase during analysis was applied as follows: 99% A/1% B at 0-1 min, 0% A/100% B at 7 min, 2% A/98% B at 8.5 min, 99% A/1% B at 10 min and 99% A/1% B at 12 min. The obtained data were analyzed using UNIFI software.
Statistical analysis. All data were expressed as mean ± SD, and statistical analysis was analyzed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test using SAS program (Ver. 9.4 SAS Institute, Cary, NC, USA). The statistical difference was presented as the different small letters (p < 0.05). www.nature.com/scientificreports/